Restricted access to human brain tissues limits the discovery of novel interventions and pharmacological treatments for one billion people with neurological disorders globally (World Health Organization, Neurological disorders affect 1 billion people: WHO Report, (World Health Org., 2007)).
Amyloid precursor protein (APP) proteolysis is fundamental for production of amyloid-b (Aβ) peptides implicated in Alzheimer's disease (AD) pathology (Golde, et al., (2000) Biochemical detection of Aβ isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer's disease. Biochim Biophys Acta 1502, 172-187; Huse & Doms, (2000) Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer's disease. Mol Neurobiol 22, 81-98; Sambamurti, et al., (2002) Advances in the cellular and molecular biology of the 1-amyloid protein in Alzheimer's disease. Neuromolecular Med 1, 1-31; Funamoto, et al., (2004) Truncated carboxyl-terminal fragments of b-amyloid precursor protein are processed to amyloid β-proteins 40 and 42. Biochemistry 43, 13532-13540). APP proteolytic products arise from the actions of α-, β-, and γ-secretases. In the amyloidogenic pathway, Aβ peptides are produced via initial action of β-secretase (BACE) cleavage, which creates an Aβ-containing carboxylterminal fragment, β-CTF or C99 (Sinha & Lieberburg, (1999) Cellular mechanisms of β-amyloid production and secretion. Proc Natl Acad Sci USA 96, 11049-11053; Yan, et al., (1999) Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537). This also generates an amino-terminal, soluble APPb (sAPPb) fragment, which is released extracellularly. Intracellularly, β-CTF is then cleaved by a multiprotein γ-secretase complex that results in generation of the Aβ peptide and a smaller γ-CTF, also known as C57 (Steiner, et al., (1999) Proteolytic processing and degradation of Alzheimer's disease relevant proteins. Biochem Soc Trans 27, 234-242). Conversely, in the anti-amyloidogenic pathway, APP is first cleaved at the asecretase site, by the putative α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10), which results in the release of amino-terminal soluble APPa (sAPPα) and the generation of α-CTF or C83 (Hooper & Turner, (2002). The search for α-secretase and its potential as a therapeutic approach to Alzheimer s disease. Curr Med Chem 9, 1107-1119).
Over the past decade, there has been intense focus on investigating the processes of APP proteolysis and Aβ production as possible targets for AD therapy (Hardy & Selkoe, (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356). Various models have been developed and proposed for their ability to accurately replicate neuronal structures and responses to these pathological events. However, the majority of the models are two dimensional and lack accurate modeling of neuronal structures and connections.
Recapitulating neural development and pathology in a three-dimensional (3-D) brain tissue model is critical for studying Alzheimer's disease (AD) progression and screening therapeutic drugs. Currently, there is no good treatment to Alzheimer's disease and the on-set of Alzheimer's disease is unclear. Recently, in vitro 3-D neural cell cultures were used to mimic brain tissue and showed high sensitivity to amyloid-β (Aβ)-induced toxicity. Compared to 2-D culture models, 3-D neural culture models promote neuronal maturation and better recapitulate Alzheimer's disease. However, the previous 3-D models were established using adult human neural stem cells and restricted access to human brain tissues limits the distribution of such models. Moreover, AD progression takes years. So, novel in vitro brain models are urgently needed for better understanding of changes in central nervous systems due to AD in a short time frame.
Human induced pluripotent stem cells (hiPSCs) emerge recently as alternative sources of primary brain cells to establish AD models in vitro. From this technology, the neural induction of hiPSCs is affected by embryonic-like extracellular matrices (eECMs). A suspension bioreactor was used to enhance the diffusion inside the spheroids, which enables complex cortical neural tissue development in vitro. The derived cortical spheroids have cortical layer-specific structure and synaptic activities. The 3-D cortical spheroids derived from hiPSCs of specific patients can retain their genetic background and thus represent patient-specific in vitro models for studying AD progression and identifying therapeutic target(s). The cellular response of the model was tested on amyloid beta (Aβ1-42) oligomer-induced neurotoxicity and evaluated the response in the presence of Wnt activator. In some variations of the cellular model, the cells are transformed using motor neuron genes, thereby allowing for modeling of motor neurons (Arlotta, et al., Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron. 2005 Jan. 20; 45(2):207-21; Hedlund, et al., Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection. Brain. 2010 August; 133(Pr 8):2313-30; Sharma, et al., Genetic and epigenetic mechanisms contribute to motor neuron pathfinding. Nature. 2000 Aug. 3; 406(6795):515-9).
Human induced pluripotent stem cells (hiPSCs) can generate allogeneic or patient-specific neural cells, cortical tissues, and even mini-brains (i.e., brain organoids), which are physiologically relevant to model neural diseases and to identify pharmacological therapeutics (Chambers, et al., Build-a-brain, Cell Stem Cell, 13 (2013) 377-378; Kinney, et al., Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics, Ann Biomed Eng, 42 (2014) 352-367; Pasca, et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture, Nat Methods, 12 (2015) 671-678; Dimos, et al., Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons, Science, 321 (2008) 1218-1221; Suzuki & Vanderhaeghen, Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells, Development, 142 (2015) 3138-3150; Schwartz, et al., Human pluripotent stem cell-derived neural constructs for predicting neural toxicity, Proc Natl Acad Sci USA, 112 (2015) 12516-12521). While some disease progressions (e.g., amyloid-β plaques) may take years, in vitro neural models derived from hiPSCs can be used to probe disease on-set and development in a shortened time frame (Kondo, et al., Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Abeta and differential drug responsiveness, Cell Stem Cell, 12 (2013) 487-496). Another advantage of in vitro models derived from hiPSCs is the ability to generate specific neuronal subtypes, which are known to exhibit differential susceptibility to disease-specific molecules (Vazin, et al., Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease, Neurobiol Dis, 62 (2014) 62-72; Mertens, et al., Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder, Nature, 527 (2015) 95-99). For example, cortical neurons derived from hiPSCs have been used to screen anti-amyloid β (Aβ) drugs and to evaluate Aβ-induced toxicity (Vazin, et al., Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease, Neurobiol Dis, 62 (2014) 62-72; Yahata, et al., Anti-Abeta drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease, PLoS One, 6 (2011) e25788; Nieweg, et al., Alzheimer's disease-related amyloid-beta induces synaptotoxicity in human iPS cell-derived neurons, Cell Death Dis, 6 (2015) e1709). Moreover, hiPSC-derived motor neurons have been derived to model a variety of motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) (Dimos, et al., Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons, Science, 321 (2008) 1218-1221; Di Giorgio, et al., Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation, Cell Stem Cell, 3 (2008) 637-648).
Generating multiple neuronal subtypes from hiPSCs with a tunable differentiation protocol to delineate differential cellular responses is in a critical medical need (Schwartz, et al., Human pluripotent stem cell-derived neural constructs for predicting neural toxicity, Proc Natl Acad Sci USA, 112 (2015) 12516-12521; Mertens, et al., Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder, Nature, 527 (2015) 95-99; Maury, et al., Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes, Nat Biotechnol, 33 (2015) 89-96; Imaizumi, et al., Controlling the regional identity of hPSC-derived neurons to uncover neuronal subtype specificity of neurological disease phenotypes, Stem Cell Reports, 5 (2015) 1010-1022). In particular, 3-D neural cultures provide a good platform to generate region-specific neuronal subtypes or human brain-like tissues (e.g., microtissues or organoids) (Pasca, et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture, Nat Methods, 12 (2015) 671-678; Schwartz, et al., Human pluripotent stem cell-derived neural constructs for predicting neural toxicity, Proc Natl Acad Sci USA, 112 (2015) 12516-12521). Compared to 2-D cultures, 3-D cultures promote neuronal cell specification and maturation, therefore better recapitulating disease pathology or predicting neural toxicity (Schwartz, et al., Human pluripotent stem cell-derived neural constructs for predicting neural toxicity, Proc Natl Acad Sci USA, 112 (2015) 12516-12521; Choi, et al., A three-dimensional human neural cell culture model of Alzheimer's disease, Nature, 515 (2014) 274-278; Zhang, et al., A 3D Alzheimer's disease culture model and the induction of P21-activated kinase mediated sensing in iPSC derived neurons, Biomaterials, 35 (2014) 1420-1428; Choi, et al., Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer's disease studies, Biomaterials, 34 (2013) 2938-2946). There are two types of 3-D cultures: scaffold-based and scaffold-free. Scaffold-based 3-D cultures use natural or synthetic scaffolds to create 3-D template that allow the cells adhere, proliferate, and differentiate. Scaffold-free 3-D cultures are based on the self-organization ability of the stem cells. The cells spontaneously organize into multicellular aggregates, spheroids, or organoids (Pasca, et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture, Nat Methods, 12 (2015) 671-678). The embryoid body (EB)-based neural differentiation is a major approach to promote the self-organization of human pluripotent stem cells (hPSCs) into complex brain-like tissue structures (Pasca, et al., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture, Nat Methods, 12 (2015) 671-678; Eiraku, et al., Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals, Cell Stem Cell, 3 (2008) 519-532; Lancaster, et al., Cerebral organoids model human brain development and microcephaly, Nature, 501 (2013) 373-379), besides the scaffold-based approaches (Hosseinkhani, et al., Engineering three-dimensional collagen-IKVAV matrix to mimic neural microenvironment, ACS Chem Neurosci, 4 (2013) 1229-1235; Abbasi, et al., Influence of oriented nanofibrous PCL scaffolds on quantitative gene expression during neural differentiation of mouse embryonic stem cells, J Biomed Mater Res A, 104 (2016) 155-164). However, functional differentiation into specific neural subtypes from hPSCs has been challenging (Nicholas, et al., Functional maturation of hPSC-derived forebrain intemeurons requires an extended timeline and mimics human neural development, Cell Stem Cell, 12 (2013) 573-586), largely because the capacity of different signaling factors that regulate 3-D neural tissue patterning in vitro has not yet been fully understood (Maury, et al., Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes, Nat Biotechnol, 33 (2015) 89-96).
In neural patterning of brain tissues, i.e., the process through which neural progenitors acquire brain regional identity, activation of sonic hedgehog (SHH) signaling induces ventral (V) identity of the developing neural ectoderm while SHH inhibition generates dorsal (D) telencephalic progenitors (i.e., affects D-V patterning) (van den Ameele, et al., Thinking out of the dish: what to learn about cortical development using pluripotent stem cells, Trends Neurosci, 37 (2014) 334-342; Vazin, et al., The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons, Biomaterials, 35 (2014) 941-948). Thus, differential levels of SHH signaling, in combination with other signaling such as Wnt and retinoic acid, influence neural regional specification of hPSCs into forebrain cortical tissues, midbrain tissues, and hindbrain/spinal cord tissues (Suzuki & Vanderhaeghen, Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells, Development, 142 (2015) 3138-3150). In biomaterials research, one attractive approach is to modulate hPSC fate decisions and differentiations using small molecules that regulate signaling pathways through defined mechanisms (Siller, et al., Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells, Stem Cell Reports, 4 (2015) 939-952; Park, et al., Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments, Biomaterials, 54 (2015) 201-212; Jiang, et al., Generation of cardiac spheres from primate pluripotent stem cells in a small molecule-based 3D system, Biomaterials, 65 (2015) 103-114; Tasnim, et al., Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules, Biomaterials, 70 (2015) 115-125). Specifically, small molecules in SHH signaling (Vazin, et al., The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons, Biomaterials, 35 (2014) 941-948; Stanton & Peng, Small-molecule modulators of the Sonic Hedgehog signaling pathway, Mol Biosyst, 6 (2010) 44-54) have been demonstrated previously to facilitate the generation of some specific neural types from hPSCs (Hu & Zhang, Differentiation of spinal motor neurons from pluripotent human stem cells, Nat Protoc, 4 (2009) 1295-1304). However, the capability of SHH-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs has not been fully investigated.
Therefore, there is an unmet need for three dimensional neuronal cell systems that can model neuronal interactions and responses to various environmental conditions, including particles and conditions that can result in neuronal damage and neuronal degeneration.